Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products

Abstract

Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC , 0.4-4.0 ng mL ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products.

Fluorescence polarisation for high‐throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay

“…Phosphodiesterase 5 (PDE5) inhibition assay scheme using fluorescence polarisation (FP) technique (adapted and modified from BPS Bioscience Inc 24 …”

“…A study has recently proposed a broad-based screening of PDE5 inhibitors in herbal dietary supplements via the PDE5 inhibition assay. 24 The assay utilised fluorescence intensity measurements of tetramethyl rhodamine-labelled cGMP in the presence of zirconyl chloride octahydrate as a quenching agent. This study, however, was not validated using real samples, and the need to measure the fluorescence intensity at seven points over a length of time may not be well-suited for high-throughput screening.…”

“…However, both of these techniques are targeted for a distinct PDE5 inhibitor or a group of PDE5 inhibitors, making them limited for broad‐based screening. A study has recently proposed a broad‐based screening of PDE5 inhibitors in herbal dietary supplements via the PDE5 inhibition assay 24 . The assay utilised fluorescence intensity measurements of tetramethyl rhodamine‐labelled cGMP in the presence of zirconyl chloride octahydrate as a quenching agent.…”

Drug adulteration of sexual enhancement supplements: a worldwide insidious public health threat

“…In addition, developing efficient methods to detect PDE5-is and any analogue whatever structure, that is based on activity, may be useful. The USA, Canada, the Czech Republic, Korea and China have developed rapid analytical tests in line with this idea [58,59,[73][74][75][76]. Thus, several supplements can be simultaneously tested and rapidly removed from the market if proven adulterated.…”

Role of Nitric Oxide in the Cardiovascular and Renal Systems

“…In most cells, the rate of synthesis of cGMP is 10-fold lower than its rate of hydrolysis by phosphodiestrase (PDE) [ 14 ]. Therefore, inhibition of PDE is considered a therapeutic target in many pathological conditions such as erectile dysfunction [ 15 ], in which an elevated levels of NO is desired. Another interaction of cGMP is with protein kinase G (PKG), which is activated upon C GMP stimulation to phosphorylate different target proteins upon cGMP stimulation.…”